Difference between revisions of "Team:CIEI-BJ/Design"

Geranyl diphosphate（GPP）can be produced through MVA or DXP pathway. Then GES can help to convert GPP to geraniol.. Geraniol will be reduced to citronellol as OYE adds hydrogen to geraniol. Researchers tried to improve efficiency of citronellol production through a MVA pathway in which GES can be expressed in engineered E. Coli to produce geraniol [1]. Some other researches also showed that certain types of OYE (OYE1 from Saccharomyces carlsbergensis [2][3], OYE2 and OYE3 from S. cerevisiae [3][4], CmOYE from Candida macedoniensis [5] and bacterial OYE homologues YqjM from Bacillus subtilis) can lead to different reaction rates for geraniol to be reduced into citronellol [6].Zeng's research showed that different OYE gene in E.coli and yeast can induce different production of citronellol [7] [8].

Our aim is to work out a pathway to take glucose as the substrate to directly produce citronellol. We will also try different composition of citronellol-based mosquito repellent to make our product has enjoyable scent and repels mosquitoes effectively. We would like to make a mosquito repellent safe enough even for babies. We will optimize the labor and resource cost to extract citronellol and make E. coli or yeast into a factory for massive production of citronellol, a harmless mosquito repellent. Therefore, we designed expression systems in both E. coli and yeast.

After mass investigation, we, CIEI-BJ, designed a brand new citronellol production procedure.

The graph below shows this process.

Fig.1 General process of synthesizing citronellol

Based on previous researches, citronellol can be produced with the catalyst old yellow enzyme (OYE) from geraniol[9], which can be synthesized by using modified E.coli with the raw material of glucose[10] . Geraniol synthase (GES) is the enzyme identified to be responsible for synthesizing geraniol. [11] In order to have a continuous production process for citronellol, we innovatively combined these two steps and utilized E.coli as the carrier for undergoing the process. We used glucose as the substrate to produce citronellol with the intermediate product geraniol. During this procedure, GES and OYE are the most important factors to determine the production of citronellol.

Thus we designed the following genetic circuits to produce the two enzymes GES and OYE, for the E.coli based automatic citronellol synthesis.

Fig.2 Our device to synthesize citronellol

In our experiment, we adopted two plasmids for two separated pathways. In the first pathway, Isopropyl β-D-Thiogalactoside (IPTG) provides activation for lacI operator during translation. 2A functions as separating GES gene and OYE gene, which can be automatic removed by cell itself. thus two independent proteins can be synthesized properly. [12]

In the second pathway, we construct two plasmids one contain GES gene, and the other contain OYE genes, the two plasmids can be transformed to one cell.

[1] TIAN Ning et al. ‘Optimization of Medium for Production of Geraniol by the Recombinant Escherichia coli’. 0253-2417(2015)04-0131-07

[2] Swiderska, M. A. et al. ‘Asymmetric bioreduction of a,b-unsaturated nitriles and ketones’. J. Mol. Catal. B Enzym. 2006, 42, 52-54

[3] Hall, M. et al. ‘Asymmetric reduction of activated alkenes using an enoate reductase from Gluconobacter oxydans’. Org. Chem.2008, 9, 1511-1516

[4] Wada, M. et al. (2003). Production of a doubly chiral compound, (4R,6R)-4-hydroxy- 2,2,6-trimethylcyclohexanone, by two-step enzymatic asymmetric reduction. Applied and Environmental Microbiology, 69, 933–937.

[5] Kataoka, M. et al. ‘Bioreduction of a,b-unsaturated ketones and aldehydes by non-conventional yeast (NCY) whole-cells’ J. Biotechnol. 2004, 114, 1-9.

[6] [7] Ying ZENG et al. ‘Identification of enzymes responsible for the reduction of geraniol to Citronellol’. DOI 10.1007/s13659-011-0032-6

[8] Gareth Norton et al.. ‘Characterisation of recombinant Hevea brasiliensis allene oxide synthase: Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity’. Plant Physiology and Biochemistry 45 (2007) 129-138.

[9] Zeng Ying et al. ‘Identification of Enzymes Responsible for the Reduction of Geraniol to Citronellol’. Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201

[10] IIJIMA Y et al. ‘Characterization of geraniol synthase from the peltate glands of sweet basil’. [J]. Plant Physiology, 2004, 134 (1): 370-379.

[11] Ning Tian. ‘Fermentation Technology for Biosynthesis of Geraniol using Engineered Escherichia coli’. May, 2015.

[12] Zhang LI et al. ‘The Study on Application of Bicistronic Vector in Transgenic Zebrafish Based on 2A Peptide’ 1000-7083(2015)03-0338-07.